Nucleus pulposus (NP) cells of the intervertebral disc are derived from notochord and synthesize a soft, gelatinous matrix containing numerous collagen, proteoglycan and laminin species. With age, NP cells become more fibroblast-like and lose their ability to synthesize and repair this NP-specific extracellular matrix. We have previously shown that environmental cues of molecular composition and substrate stiffness can be manipulated to promote elevated matrix synthesis for the NP cell. In particular, we have developed a laminin-presenting hydrogel that can promote increased expression for many molecular markers of the healthy, biosynthetically active NP cell (e.g, brachyury, integrin ?3, laminin, and elevated sGAG), but only when crosslinked to be soft (<0.5 kPa) as opposed to stiff (>0.9 kPa). Our overall hypothesis is that environmental cues of soft stiffness and laminin engagement can be exploited to promote re-expression of the healthy, biosynthetically active NP cell phenotype for cells of the adult, degenerate human NP. In Specific Aim 1, we will determine if young and/or adult degenerate human NP cells have elevated expression of NP-specific molecular markers, elevated NP-specific matrix biosynthesis, and NP-related transcription factor activity following culture upon laminin-functionalized polyethylene (PEG) hydrogels of varying stiffness (0.3 -50 kPa). NP-specific markers will be measured with mRNA, protein and biochemical assays following guidance from a consensus paper by the Spine Research Interest Group (Risbud et al. 2015, JOR). In Specific Aim 2, we will determine if cell recognition peptides have an ability to promote elevated expression of NP-specific markers for young and/or adult degenerate human NP cells when cultured upon peptide-functionalized PEG hydrogels. We have identified four cell recognition peptides derived from integrin-binding and laminin-derived domains that promote human NP cell attachment and elevated sGAG synthesis when attached to soft hydrogel substrates. We will vary ratios of these four peptides upon PEG substrates of optimal stiffness identified from Specific Aim 1, and measure the expression of NP-specific molecular markers over time (as for Specific Aim 1). In Specific Aim 3, we test if an optimal PEG-laminin or PEG-peptide hydrogel can preserve the re-expression of the healthy NP cell phenotype for degenerate human NP cells following delivery to the pathological environment of the degenerated intervertebral disc. Adult, degenerate NP cells will be preconditioned upon PEG-laminin or PEG- peptide substrates, then delivered into degenerated discs in a nude rat model with an injectable, in situ crosslinking version of the PEG hydrogel as a cell carrier. Cell residence time (luminescence), disc height, and NP-specific markers will be evaluated to test if the PEG-LM or PEG-peptide hydrogel can support preservation of the healthy NP cell phenotype for these degenerate NP cells in the native disc. Completion of this study would identify environmental cues that can promote re-expression of a healthy, biosynthetically active NP cell with application to primary adult NP cells and progenitor cells for cell-mediated regeneration of the degenerate disc.